Polymers consist of chains of repeat chemical units that occur naturally or by the virtue of their chemistry, they can be adapted to form synthetic polymers by control of the chemical structure and weight.
Synthetic polymers are made by the chemical reaction of monomers to form long polymer chains. The two primary variables that affect the physical properties of synthetic polymers are the chemical nature of the monomer repeat units and the molecular weight of the polymer, which can be precisely controlled. Thus, it is known that polymers with higher molecular weights possess greater mechanical strength, but are much more viscous in solution.
By varying the ratio of chemical groups within the polymer, one can adjust the physical properties of the polymer for particular applications. For instance, the mechanical strength and solubility of the polymers can be adjusted in this manner.
By blending different types of polymers, characteristics of the finished copolymer can exhibit optimized characteristics. By design and synthesis of specific polymers, materials can be generated with the exact properties required for a specific function. For example, by specific treatment, the surface area of a polymer plate (90 mm) can be increased from 1 meter squared to 1,500 meter squared. This single process will increase the physical and biological characteristics of the polymer into a more efficient mechanism for recovery of environmental organisms.
Water-soluble polymers are known in the art and are described by Prokop et al in “Water Soluble Polymers for Immunoisolation I: Complex Coacervation and Cytotoxicity” in Advances in Polymer Science, 136: pgs. 53 to 73 (1998). These polymers have a multitude of uses such as artificial hearts, as immunoisolation barriers, for pain control for terminal cancer patients and in the encapsulation of pancreatic islets.
Besides their medical uses, polymeric material is also used in laboratories in many of the supplies such as tests tubes, sample wells, pipette tips, disposable pipettes and the like.
For instance, as a solid support, various polymers were used in a method for detecting DNA in a cell, while preserving the morphology of the nucleus as described in U.S. Pat. No. 5,501,954. In this method the DNA was deposited onto a polymeric membrane filter, incubated with a fluorescently labeled sample and detected using a labeled probe. The polymeric membranes utilized were made of polycarbonate, polyvinylidene fluoride, polysulfone, nylon, cellulosic esters, nitrocellulose and Teflon® (PTFE). The polymeric membranes described in this patent are water-insoluble polymers, since one of the requirements for this method is that the polymeric membranes must retain the cellular material through a series of treatments and washings and still remain intact.
EP 546 032 describes a method for immobilizing molecules, polymers or microorganisms by mixing with an aqueous solution dispersion of a polymer and applying the mixture to a coherent film. The membranes formed are water insoluble and can be stored dry.
The analysis of microorganisms is important in many different areas, for instance, in food preparation, drinking water, for pharmaceutical applications in drug production, for cosmetic analysis, in electronic industries and in the analysis of medical applications.
Samples for the testing of various microorganisms are generally collected using cotton swabs, for example, and sent to a laboratory for analysis. The analysis process requires that the samples first are cultured.
Alternatively rapid methods for analysis of microorganisms are also known in the art using biosensors. For example, WO 9931486 discloses biosensors having a polymer film coated with a metal and a patterned receptor layer printed onto the coated metal on which there is a receptive material that specifically binds analyte. The amount of microorganism that was attached to the biosensor was measured via a diffraction image upon irradiation with a laser.
Another type of biosensor is disclosed in WO 982747 which comprises a polymer film coated with a metal and a self-assembling monolayer having a receptive material on it specific for an analyte, printed onto the film. The self-assembling monolayer is printed in a pattern, so that, when the biosensor binds the analyte and the biosensor diffracts transmitted light to form a pattern.
It should be appreciated that although biosensors can detect various microorganisms in the environment measuring a diffraction pattern is often inaccurate, imprecise and lacks sensitivity.
Moreover, determination of the number of active microorganisms rather than total counts is of great importance in many areas of microbiology. Unfortunately, it is widely recognized that conventional culture techniques underestimate the fraction of, true viable microorganisms and that total counts, showing all microorganism particles, overestimate this fraction.
Besides the problems associated with obtaining an accurate number of microorganisms present in a sample, it is also known that culturing techniques on a growth medium are time-consuming and generally require between about eighteen hours and twenty days to obtain a result. The use of traditional growth medium is non-specific and natural, therefore variable and non-controlled.
One method that overcomes the requirement for the culturing of microorganisms after they are sampled is described in EP 0 816 513A1. This reference discloses the use of a pressure sensitive adhesive sheet for collecting microorganisms on surfaces that may contain the microorganism. The adhesive sheet is composed of a laminate of an adhesive layer mainly composed of a water soluble polymer and a water permeable membrane which does not permit the passage of microorganisms. Hence, EP 0 816 513A1 requires that at least two layers of the adhesive sheet be bonded together, one layer of which acts to capture the microorganisms in a process after sampling.
Moreover, the sampling of the microorganism with the pressure sensitive adhesive sheet requires that the adhesive layer be brought in contact with the surface of a test object such that accumulation of microorganisms is accomplished on the sheet Hence, even visual observation of microorganisms is accomplished using a chromagenic agent which is present in the adhesive layer or in water, which method cannot be very sensitive.
EP 0 816 513 A1 does not disclose or suggest that either their device to sample, requiring at least two laminated layers, and/or their method to detect microorganisms can be used for sterility testing of air samples in which very high sensitivity of detection is required.
Indeed, it is well known in the art that specific monitoring and control of aseptic environments is required for the processing of drugs, dosage forms and in certain cases medical devices. A large portion of sterile products are manufactured by aseptic processing since this process relies on the exclusion of microorganisms from the process stream and the prevention of microorganisms from entering containers during filling. Aseptic processing is generally performed in clean rooms and the environment is always carefully monitored.
Besides the use of aseptic conditions in the pharmaceutical industry, the electronic industry also uses and monitors clean rooms for the manufacture of electronic components, computer chips, computer components and the like.
The difference between these two industries in environmental monitoring is that in the electronic industry nonviable microorganisms or particulates are generally measured and there is less emphasis on the number of viable particulates or microorganisms. In contrast, in the pharmaceutical industry there is a much greater concern with respect to the point of viable microorganisms.
One method of monitoring in aseptic conditions is to ascertain the total particulate count. This method does not provide information concerning the microbiological content of the environment. The basic limitation of particulate counters is that they only measure particles of 0.5 μm or larger. While airborne particles are not free-floating or single cells, they frequently associate with particles of 10 μm to 20 μm and hence solely testing for particulate counts without microbial counts is discouraged.
It is known in the art that clean rooms must meet particular standards. In fact specifications for air changes per hour and velocities, although not included in federal standards, are customary. Thus, for example class 100,000 rooms in aseptic processing environments are designed to provide a minimum of 20 air changes per hour, while class 100 clean rooms provide more than 100 air changes per hour. By diluting and removing contaminants, large volumes of air are likely to reduce airborne contamination in aseptic production.
There are certain air cleanliness guidelines that must be met for the different grades of a clean room. Thus, for example, for class 100, the running mean of all data points must be <1 colony-forming unit (cfu) per cubic meter of air and at least 85% of all samples taken must be zero. For class 10,000 clean rooms, at least 65% of all the samples taken must be zero and for class 100,000 at least 50% of samples must be zero. Thus, these values are very critical in order to provide safe environmental monitoring.
There are many methods known in the art to sample viable airborne microorganisms such as the slit-to-agar sampler, the sieve impactor, the centrifugal sampler the surface air system sampler and the gelatin filter sampler. All of these samplers require a pump, motor or vacuum that either pulls or pushes air through the sampling unit The use of these “active” sampling devices can be inconvenient where there is space limitation in the clean room since they may occupy needed space. Moreover, these devices may also be a hazard to safe aseptic conditions, since they can disrupt directional air flow as a result of the size and location of the instrument or of the manner in which the equipment forces air into the sampling media or filter.
Another type of “non-active” sampling devices is settling plates. Setting plates are an easy and inexpensive way to qualitatively assess the air environment over long periods of time. Settling plates consist of agar which are placed in Petri dishes and are useful in critical areas where the use of an active sampling device is obstructive. In fact settling plates, when exposed for four to five hour periods, may provide a limit of detection similar to those observed with active sampling devices.
However, in many of these methods, agar is used as the medium to capture the microorganisms and it is known that agar shortages, as well as product variability have led to a search for suitable substitutes for agar.
Furthermore, it is well known in the art that the monitoring of microorganisms under aseptic conditions is not as yet perfected. Variations in sampling sensitivity and limits of detection can be attributed not only to the inherent characteristics of the sampling method itself, but also to media variability, incubation temperatures, sample handling and accidental contamination of the samples.
Moreover, microbial assessment of dean rooms is performed using methods that do not result in a quantitative assessment Rather, the methods used can be at best defined as semiquantitative. In fact many methods are only suitable to measure the presence of a typically high levels of microbial contamination and their accuracy and precision is very poor.
Therefore, there is a need in the art of environmental analysis of microorganisms to provide not only a rapid method of analysis for air samples, but also a means to accomplish this analysis with greater accuracy, especially in the sterility testing area.
Thus, it is an object of the present invention to overcome the problems associated with the prior art.
It is an object of the present invention to use water-soluble polymers to trap and confine microorganisms in air.
It is another object of the present invention to provide a sensitive means for sterility analysis of air samples.
It is another object of the present invention to provide a polymer that has sufficient mechanical strength so that it can be transported, is soluble in water or other physiological diluents, retains water to a certain degree while maintaining live microorganisms in a viable status and can capture microorganisms from the air.
It is yet another object of the present invention to provide a process for rapidly detecting microorganisms in an unknown air sample.
It is a further object of the present invention to provide a process to detect microorganisms in an unknown air sample which does not require that the microorganism be subjected to further growth.
It is another object of the present invention to provide a process in which the variable and uncontrolled system of using growth medium is avoided.
These and other objects are achieved by the present invention as evidenced by the summary of the invention, description of the preferred embodiments and the claims.